Method of detecting target nucleic acid molecules
Assignee10x Genomics, Inc.
InventorsMalte Kühnemund, Toon Verheyen
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Abstract
The present application provides methods for detecting a nucleic acid molecule involving the use of a signal code sequence which corresponds to said nucleic acid molecule and a plurality of labelled detection probes which yield signals which make up the signal code sequence. In particular, the invention provides a sequential barcoding and decoding scheme which utilises a sequencing-by-hybridisation (SBH) strategy to sequence and decode a nucleotide barcode sequence, and to differentiate the nucleotide barcode sequence from other nucleotide barcode sequences. In an extension of the method, the application also provides a new coding scheme for providing a target nucleic acid with a detectable “colour” (or similar signal)-based code.
Claims17
A method of detecting a target nucleic acid molecule in a sample, comprising:
- (a) contacting the sample with a padlock probe specific for the target molecule, wherein the padlock probe is circularized after hybridization to a target sequence in the target nucleic acid molecule, wherein the padlock probe comprises a nucleotide barcode sequence to identify the padlock probe, and wherein the circularized padlock probe is amplified by rolling circle amplification (RCA) to produce a rolling circle product (RCP), the RCP containing multiple complementary copies of the nucleotide barcode sequence;
- (b) assigning a signal code sequence specific to the target nucleic acid molecule,
- wherein the signal code sequence may be derived by interrogating the complementary copies of the nucleotide barcode sequence with sequential detection probes each yielding a signal and the signals together make up the signal code sequence, and wherein a complementary copy of the nucleotide barcode sequence comprises a first domain and a second domain, wherein a portion of the first domain overlaps with some but not all of the second domain;
- (c) contacting the RCP with a first detection probe to hybridize the first detection probe to the RCP, wherein the first detection probe comprises:
- (i) a recognition sequence complementary to the first domain and
- (ii) a first reporter;
- (d) detecting a signal from the first reporter, thereby obtaining a first signal code of the signal code sequence;
- (e) contacting the RCP with a second detection probe to hybridize the second detection probe to the RCP, wherein the second detection probe comprises:
- (i) a recognition sequence complementary to the second domain and
- (ii) a second reporter,
- wherein the second detection probe initiates a strand displacement reaction displacing the first detection probe hybridized to the first domain;
- (f) detecting a signal from the second reporter, thereby obtaining a second signal code of the signal code sequence;
- and
- (g) contacting the RCP with additional detection probes to identify additional signal codes of the signal code sequence until sufficient signal codes have been identified to detect the nucleic acid molecule, wherein the additional detection probes have recognition sequences complementary to the first or second domain such that additional detection probes successively hybridize to the first or second domain and displace preceding detection probes hybridized thereto.
Dependencies are inferred from the claim text.